Preparation of parental cell bank from foetal tissue

ABSTRACT

The present invention relates to methods of in vitro preparation of a parental cell bank (PCB) from foetal tissue consisting of foetal epiphyseal tissue, foetal Achilles tendon tissue and foetal skin tissue, using a rapid mechanical primary cell culture selection of cell type to be used in methods for wound and tissue repair.

PRIORITY STATEMENT

This application is a national stage application under 35 U.S.C. §371 ofPCT International Application No. PCT/IB2012/053512 which has anInternational filing date of 10 Jul. 2012 and claims priority under 35U.S.C. §119 to European Application No. 11173452.1 filed 11 Jul. 2011.The contents of each application recited above are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods of in vitro preparation of aparental cell bank (PCB) from foetal tissue consisting of foetalepiphyseal tissue, foetal Achilles tendon tissue and foetal skin tissue,using a rapid mechanical primary cell culture selection of cell type tobe used in methods for wound and tissue repair.

BACKGROUND OF THE INVENTION

Cellular therapy is becoming an interesting addition for medicaltherapies for repairing, restoring or ameliorating function of tissuesand particularly combining with traditional surgical techniques. Somecell choices are more adaptable to cellular therapy in patients. Tissuechoices from animal and human at all ages of development can beevaluated with advantages and disadvantages for each final cell type.Current restrictions for human cell-based therapies have been related totechnological limitations with regards to cellular proliferationcapacity (simple culture conditions), maintenance of differentiatedphenotype for primary human cell culture, transmission of communicablediseases and the consistency and stability of the selected population ofcells depending on their isolation procedure. Cultured primary foetalcells from one organ donation meet the exigent and stringent technicalaspects for development of therapeutic products. Master and working cellbanks from one foetal organ donation can be developed in short periodsof time and safety tests can be performed at all stages of cell banking.

Cell therapy has been proposed as a less invasive alternative orcombined therapy for surgical procedures and tissue engineering ofspecific tissues. Several cell types have been investigated to beutilized in cell therapy: embryonic stem cells (ES), umbilical cordcells, foetal cells and adult stem cells (from bone marrow-haematopoiticstem cells or HSCs and marrow stem cells or MSCs) along with adiposetissue, platelets, placenta and amniotic fluid cells. As for anyapplication in tissue engineering, the cell origin and type areessential aspects. Each type of cell requires different methods tomanipulate their differentiation and self-renewal capabilities forspecific therapies with various advantages and disadvantages.

Foetal cells have been used extensively in biology and medicine for manyyears without much public knowledge for their importance, especially inthe development of necessary vaccines. Foetal cells are differentiatedcells with high expansion, regeneration and low immunogenic properties.They can be isolated from foetal tissues, which follow embryonic stageafter 9 weeks of development. Foetal skin cells offer an ideal solutionfor effective and safe cell therapy and tissue engineering for severalreasons including; a) cell expansion capacity from one organ donation;b) minimal cell growth requirements; c) adaptation to biomaterials fordelivery; and, d) resistance to oxidative stress. Foetal skin cells haveextensive expansion possibilities as it requires only one organ donation(1-4 cm² tissue) to create enough frozen cells to produce a bank capableof hundreds of thousands of treatments (i.e. for skin, over 35 billionfetal skin constructs 9×12 cm, can be produced from one dedicated cellbank). Also, cell culture requirements are minimal compared to stem ormesenchymal cell types. As the foetal skin cells are alreadydifferentiated and do not need to be directed or altered, the vastnumber of additional growth factors normally necessary are not neededfor cell culture and expansion. For cell banking, careful selection of adonor and an extensive screening of both the donor and cultured cells toavoid transmissible viral, fungal or bacterial disease provide a safeand secure utilization of foetal cells for therapeutic usage. Inaddition, foetal cells, unlike neonatal, young or adult cells adaptparticularly well to biomaterials allowing efficient and simple deliveryto the patient. It has been shown that cells from donors (neonatal toadult) are not capable of efficient integration into variousbiomaterials and some biomaterials are, in fact, toxic to the cell. Itis true that the scaffold is very important for tissue engineering, butthe cell type is most probably the limiting factor. For processing of afinal product for clinical delivery, both the homologous distributionand the rapidity of development of the final product are majorsignificant advantages. When long culture periods are necessary as forautologous grafting or for the commercially available products to date,there is a non-negligible increased risk for contamination. It is alsoimportant to have a process that is consistent and easily repeated. Bydeveloping consistent cell banks with fetal cells, many of the riskfactors can be eliminated for bringing safe and effective humancell-based therapies to the bedside.

Key elements including identity, purity, sterility, stability, safetyand efficacy are recommended for cellular-based products. In all, thenew regulations impose strict criteria for the production and theenvironment used for the production of cell-based products to be used inclinical trials and treatments. Current restrictions for humancell-based therapies have been related to technological limitations withregards to cellular proliferation capacity (simple culture conditions),maintenance of differentiated phenotype for primary human cell culture,transmission of communicable diseases and the consistency and stabilityof the selected population of cells depending on their isolationprocedure. Cultured primary foetal cells from one organ donation meetthe exigent and stringent technical aspects for development oftherapeutic products. Master and working cell banks from one foetalorgan donation can be developed in short periods of time and safetytests can be performed at all stages of cell banking. For therapeuticuse, foetal cells can be used up to two thirds of their life-span in anout-scaling process and consistency for several biological propertiesincludes protein concentration, gene expression and biological activityare ensured.

The relatively simple manipulation of foetal cells, related to theircollection, culture expansion and storage has made foetal cells anattractive choice for cell therapy. Unlike ES cells, foetal cells do notform tumours and seem to lack immunogenecity when transplanted. Incontrast with mesenchymal stem cells to date, foetal cells do notrequire feeder layers for growth or specific growth factors fordifferentiation. One organ donation is capable of producing aconsistent, Master Cell Bank (MCB) that would be available for hundredsof thousands of patient treatments. The fully-defined consistent cellbank could easily be assessed for safety concerning any potential virusand pathogens in parallel to the original organ donation where serologyand pathology are accomplished.

Primary cultures of foetal differentiated cells from specific tissuessuch as cartilage, tendon and skin which specific cell sources can bedeveloped including chondrocytes (chondro-progenitors), skin fibroblastprogenitors, and tendon progenitors determine the quality of theclinical cell bank developed. It is well accepted that the initialtreatment of the tissue is of major importance to the physiologicalproperties of the cells thereafter produced. The routinestate-of-the-art in primary culture is to enzymatically digest the smallpieces of tissue to liberate all live cells for cell culture. This isparticularly used for “hard” tissues, but also for soft tissues, wheremultiple digestion steps are used routinely. By doing this, a completelydifferent population of cells is liberated and cultured in the primaryand secondary cell cultures (Carrascosa A, Audi L, Ballabriga APediatric Research 19:720-727, 1985; Roche S et al, Biomaterials22:9-18, 2001; Bae H. et al., The Spine Journal Vol. 8, No. 5, pages92S-93S, 2008; Reginato A. M. et al., Arthritis and Rheumatism, Vol. 37,No. 9, 1994).

However, it is also known that the enzymatic treatment causesinconsistencies, develops populations of cells with differentmorphologies, physiological properties, stability, and function(Diaz-Romero J, Gaillard J-P, Grogan P, Nesic, Trub T, Varlet P-M JCellular Physiol 202:731-742, 2005).

The avascular, aneural and alymphatic nature of articular cartilage hasmade repair of this tissue a challenge for both surgeons and tissueengineers. A gold standard for osteochondral therapeutic strategies,especially for the choice of cell source remaining a central andcontroversial issue, is far from being determined. Adult mesenchymalstromal cells (MSCs) are, to date, the most used cell source, despiteconcerns of phenotypic homogeneity, reliability and stability.

Treatment of osteoarthritic defects needs to be improved as nosatisfactory therapeutic solution exists to date. It is especiallycrucial to develop new solutions to avoid premature degeneration of thecartilage to avoid total joint replacement. Development of new cellularassisted surgical techniques is based on a defined cellular bankedproduct that can meet the requirements for stringent therapeutic agentpreparation.

Thus there is still a need to develop methods for producing new tissuessuch as tendon, cartilage, other musculoskeletal tissues, and skin, foruse in the therapeutic strategies. More specifically there is a need todevelop cell banks which provide more stable and uniform population ofcells and to find an appropriate source of cells, that do not risktriggering an immune response, and that do not carry any infectiousagents.

SUMMARY OF THE INVENTION

To solve the above-identified problem, Applicants have established anon-enzymatic method to liberate mechanically and rapidly early adherentcell populations that define the characteristics of a parental cell bank(PCB) establishment. In some embodiments, the primary, differentiatedcells come from specific cartilage tissue, specific tendon tissue andspecific skin tissue. To one skilled in the art, other embodiments couldinclude primary differentiated cells from skin and musculoskeletaltissues such as, tendon, bone, muscle and vertebral disc. Thedevelopment of the PCB allows consistent and stable further cell bankingto be accomplished.

Specifically in one embodiment the present invention provides an invitro non-enzymatic method for isolation, expansion and development offoetal cells selected from the group consisting of foetal epiphysealchondrocytes, foetal Achilles tenocytes or foetal skin fibroblsts,comprising the steps of:

-   -   a) using foetal sample selected from ulnar foetal cartilage        comprising foetal epiphyseal chondrocyte; foetal Achilles tendon        comprising foetal Achilles tenocytes; or foetal abdominal skin        comprising foetal skin fibroblasts;    -   b) micro-dissecting and dispersing said ulnar foetal cartilage,        foetal Achilles tendon or foetal abdominal skin sample by        mechanical attachment to scalpel scored surface;    -   c) culturing said ulnar foetal cartilage, foetal Achilles tendon        or foetal abdominal skin sample in vitro under conditions        wherein said foetal epiphyseal chondrocytes, foetal Achilles        tenocytes or foetal abdominal skin fibroblasts proliferate,    -   d) selecting and isolating first adherent foetal epiphyseal        chondrocyte cell populations, first adherent foetal Achilles        tenocyte cell populations and first adherent foetal skin        fibroblast populations therefrom.

In another embodiment, the present invention provides foetal cellsobtained by the non-enzymatic method of the invention, such as foetalepiphyseal chondrocyte cell line having the designation FE002-Cart anddeposited under accession number ECACC 12070303-FE002-Cart, foetalAchilles tenocyte cell line having the designation FE002-Ten anddeposited under accession number ECACC 12070302-FE002-Ten and foetalskin fibroblast cell line having the designation FE002-SK2 and depositedunder accession number ECACC 12070301-SK2.

In another embodiment, the present invention provides a use of foetalcells obtained by the method of the present invention for the productionof new cartilage tissue and/or three dimensional constructs, new tendontissue and/or three dimensional constructs, new skin tissue and/or threedimensional constructs, by using the differentiated cartilage, tendon orskin cells integrated into various matrixes.

In another embodiment, the present invention provides the foetal cellsobtained by the method of the present invention for use as therapeuticagent.

In another embodiment, the present invention provides foetal cellsobtained by the method of the present invention for use in a method forrepair and regeneration of osteochondral tissue and musculoskeletaltissue, for use in a method for repair and regeneration of tendon tissueand musculoskeletal tissue, and for use in a method for repair andregeneration of skin tissue and for treating burns, wounds and fibroticcondition.

In another embodiment, the present invention provides foetal cellsobtained by the method of the present invention for use in a method fortreating osteochondral diseases or defects, arthritis andmusculoskeletal diseases; for use in a method for treatingmusculoskeletal diseases and tendonopathies; for use in a method fortreating skin diseases.

In further embodiment of the present invention is provided a screeningmethod for development of therapeutic agents and/or medical devices forthe treatment of arthritic, osteochondral defects, cartilage repair,tendon repair, musculoskeletal tissue repair and skin repair, comprisingthe use of foetal epiphyseal chondrocyte (FEC), foetal Achillestenocytes or foetal skin fibroblasts obtained by the non-enzymaticmethod of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows tissue biopsy to begin Parental Cell Bank production forfoetal skin progenitors and cell selection at Day 4 showing consistency.

FIG. 2 shows cell growth of foetal skin progenitors following lowdensity seeding (˜2000 cells/cm²) and recovery of frozen cell stocks atDay 6 and 12 showing high stability, consistency and maintainedfunction.

FIG. 3 shows morphological and growth differences of cell populationselection if tissue is enzymatically digested to prepare Parental CellBanks and non-consistency of cell population.

FIG. 4 shows tissue biopsy to begin Parental Cell Bank production forfoetal tendon progenitors (foetal Achilles tenocytes) and cell selectionat Day 4 showing consistency.

FIG. 5 shows cell growth of foetal tendon progenitors following lowdensity seeding (˜2000 cells/cm²) and recovery of frozen cell stocks atDay 0, 3, 7 and 10 showing high stability, consistency and maintainedfunction.

FIG. 6 shows FACS analysis and 3D matrix deposition characteristics forfoetal tendon progenitors.

FIG. 7 shows processing of proximal ulnar epiphysis tissue to beginParental Cell Bank production for foetal chondro-progenitors (foetalepiphyseal chondrocytes) and cell selection at Day 6 showingconsistency.

FIG. 8 shows cell growth of foetal chondro-progenitors (foetalepiphyseal chondrocytes) following low density seeding (˜2000 cells/cm²)and recovery of frozen cell stocks at Day 6 and 12 showing highstability, consistency and maintained function.

FIG. 9 shows FACS analysis and 3D matrix deposition characteristics forfoetal chondro-progenitors (foetal epiphyseal chondrocytes).

FIG. 10 shows foetal cell progenitor cell expansion on tissue cultureplastic directed along mechanical scored-plated.

FIG. 11 shows rapid development of clinical parental cell banks usingnon-enzymatic preparation of tissue.

DETAILED DESCRIPTION OF THE INVENTION

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. The publications andapplications discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention. Inaddition, the materials, methods, and examples are illustrative only andare not intended to be limiting.

In the case of conflict, the present specification, includingdefinitions, will control. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as is commonlyunderstood by one of skill in art to which the subject matter hereinbelongs. As used herein, the following definitions are supplied in orderto facilitate the understanding of the present invention.

As herein used, “a” or “an” means “at least one” or “one or more.”

The term “comprise” is generally used in the sense of include, that isto say permitting the presence of one or more features or components.

The term “biomaterial” means a natural or synthetic material, includingmetals, ceramics and polymers devoid of deleterious effects when incontact with cells or biological tissues. Usually, the biomaterialsupport is selected from the group consisting of polymeric supportcomprising olefin polymers, fluorine polymers, polystyrene, polyacrylicpolymers, polyesters polymers, polyurethane polymers, silicon polymers,cellulose polymers, epoxy polymers, silicone-based polymers, synthetichydrogels, polycarbonates; biocompatible metallic supports comprisingtitanium and titanium alloys, nitinol, zirconia, stainless steel andcobalt chromium, alumina-zirconia composites; and/or biocompatibleceramics comprising porcelain, hydroxyapatite, and mixtures thereof.

The term “foetal chondrocyte or chondro-progenitor”, “foetal Achillestenocyte or foetal tendon progenitor” or “foetal fibroblast or foetalskin progenitor” means differentiated cells compared to undifferentiatedfoetal cells. Contrary to the present invention, the term“undifferentiated” is used to describe an immature or primitive cell.For example, undifferentiated foetal skin cells include those that candifferentiate into dermal fibroblasts and epidermal keratinocytes oreven other specific cell types of unrelated tissue. Differentiated cellsare those that when placed in differentiation media specific for anothercell type or into a different micro-environment, will notde-differentiate into a different cell lineage. For instance, if foetalskin fibroblasts are placed into osteogenic differentiation media, theywill not become a whole population of osteoblasts or if they are placedin adipogenic media they will not become a whole population ofadipocytes and if the same cells are placed into a 3D matrix inassociation with bone, will not de-differentiate into a whole populationof osteoblasts because of the change in environment. These defined,differentiated cells have then major advantages for their potential useas therapeutic agents for both human and veterinary medicine.

The term “appropriate culture conditions” or “conditions in which foetalepiphyseal chondrocytes or chondro-progenitors, foetal tendonprogenitors or foetal skin progenitors proliferate” is a medium forculturing cells containing nutrients that promote proliferation. Thenutrient medium may contain any of the following in an appropriatecombination and in the appropriate concentrations: isotonic saline,buffer, amino acids, serum or serum replacement, and other exogenouslyadded factors. Those skilled in the art will recognize that any commonlyemployed culture conditions can be used. Methods for the selection ofthe most appropriate culture medium, medium preparation, and cellculture techniques are well known in the art and are described in avariety of sources, including Doyle et al., (eds.), 1995, Cell & TissueCulture: Laboratory Procedures, John Wiley & Sons, Chichester; and Hoand Wang (eds.), 1991, Animal Cell Bioreactors, Butterworth-Heinemann,Boston, which are incorporated herein by reference. For example anyappropriate type of culture medium can be used to isolate the foetalepiphyseal chondrocytes of the invention, such as, but not limited to,DMEM, McCoys 5A medium (Gibco), Eagle's basal medium, CMRL medium,Glasgow minimum essential medium, Ham's F-12 medium, Iscove's modifiedDulbecco's medium, Liebovitz' L-15 medium, and RPMI 1640, serum-freemedia among others. The culture medium may be supplemented with one ormore components including, for example, fetal bovine serum (FBS),defined growth factor serum replacements, equine serum (ES), HUMAN SERUM(HS), defined cell culture growth factors and one or more antibioticsand/or antimycotics to control microbial contamination, such as, forexample, penicillin G, streptomycin sulfate, amphotericin B, gentamicin,and nystatin, either alone or in combination, among others.

The term “cell line” refers to a permanently established cell culturethat will proliferate indefinitely given appropriate fresh medium andsufficient space. The term primary cell line refers to an establishedcell culture with limited passage numbers.

The term “cell bank” refers to harvesting biopsies from donor foetaltissue, such as ulnar foetal cartilage, foetal Achilles tendon or foetalskin; growing the foetal tissue and proliferating foetal cells to a highconcentration under appropriate culture conditions; using enzymatic ornon-enymatic treatments (i.e. trypsin) to the tissue and cells of theresulting cultures to allow their suspension; pooling the suspendedcells to make a generally uniform suspension of cells from the culture;gently mixing with a cryoprotectant; sealing aliquots of the cellsuspension in ampoules; and freezing the aliquots. The optimal rate offreezing may be determined empirically. For example by decreasing thetemperature of the ampoule by 1° C./min until −80° C. and thentransferred to −160° C. approximately 24 hours later, or in programmedcycles in a automatic, calibrated Nano-Freezer for full cycle freezingto −165° C. This ultra-cold temperature bank preserves the cells suchthat they stop aging, thereby allowing them to retain the function andactivity they had on the day they were collected.

The cryopreserved cells of the invention constitute a cell bank(1-10⁷/ml), portions of which can be “withdrawn” by thawing and thenused to produce new cartilage cells and tissue as needed. Thawing shouldgenerally be carried out rapidly, for example, by transferring anampoule from liquid nitrogen to a 37° C. water bath. The thawed contentsof the ampoule should be immediately transferred under sterileconditions to a culture vessel containing an appropriate medium such asDMEM conditioned with 10% FBS. It is advisable that the cells in theculture medium be adjusted to an initial density of about 1-6−10³⁻⁴cells/ml. Once in culture, the cells may be examined daily, for example,with an inverted microscope to detect cell proliferation, andsubcultured as soon as they reach an appropriate density or be monitoredin real-time with scanning microscopy for quality control.

The cells of the invention may be withdrawn from the bank as needed, andused for the production of new cartilage, tendon or skin tissue or cellseither in vitro, for example, as a three dimensional cartilage, tendonor skin culture, as described herein, or in vivo, for example, by directadministration of cells to the site in a subject where new cartilage,new tendon or new skin tissue or cells are needed.

The term “subject” (as in treatment of “a subject”) or “patient” isintended to refer to a mammalian individual afflicted with, prone to, orsuffering a condition, defect, disorder or disease (as specifiedherein). The term does not denote a particular age or sex. Thus, adultand newborn subjects, whether male or female, are intended to becovered. This term also includes both humans and animals. For example,the subjects can be, e.g., humans, non-human primates, wildlife, dogs,cats, horses, cows, pigs, sheep, goats, rabbits, rats, or mice.Preferably subjects are humans and horses. As used herein, the termwildlife includes any mammals, birds, amphibians or fish that are notdomesticated. Examples of such wildlife include, but are not limited to,badgers, beavers, lions, tigers, bears, hawks and deer.

A three dimensional matrix means any matrix selected from a collagenmatrix or PLA, PLGA, PEG, chitosan, elastin, hydrogel including forexample HA (hyaluronic acid), silicone, chitosan or a mixture thereof.The matrix provides a three dimensional space to assure proper coverageand delivery of foetal cells, such as foetal epiphyseal chondrocytes orchondro-progenitors, foetal tendon progenitors, foetal skin progenitors,or foetal products to or also in association with an additional implantmaterial. In one embodiment, the method of the invention allowspreparation of three dimensional constructs using the differentiatedcartilage, tendon or skin cells integrated into various matrixes. Theintegration of the differentiated cartilage, tendon or skin cells withmatrix can occur by mixing, combining, pipetting, seeding, plating orplacing the cells within a matrix.

The term “collagen” refers to a polypeptide compound, which ishydrophilic in nature that is subject to degradation by extracellularenzymes. Because, this substance is well studied, many key parameterscan be controlled. Collagen is a weak antigen, thereby resulting inminimal rejection potential. A preferred collagen used in the methodsand uses of the invention is horse collagen or porcine collagen.

An “implant” can be considered as a medical device that is to replace amissing biological structure, support a damaged biological structure, orenhance an existing biological structure. Implants can be made ofartificial or natural materials. Medical implants are man-made devicesand the surface of implants that contact the body might be made of abiomedical material such as titanium, silicone, polymers, apatite,biofoams and biogels. Some implants can have associated bioactiveeluting drugs such as implantable capsules or drug-eluting stents.Implant materials can be associated with specific tissue-typedifferentiated foetal cells for anti-fibrotic response.

The terms “delivery systems” mean any metallic or regularly usedorthopedic, trauma, maxillo-facial natural or synthetic implantmaterials, hydrogels, silicones or grafts providing a means to transferfoetal cells or foetal cell products alone or in association with animplant to treat tissue or render implant.

The term “cartilage tissue” is used herein as that term is generallyrecognized in the art, and refers to a specialized type of denseconnective tissue comprising cells embedded in an ECM (see, for example,Cormack, 1987, Ham's Histology, 9th Ed., J. B. Lippincott Co., pp.266-272). The biochemical composition of cartilage differs according totype; however, the general composition of cartilage compriseschondrocytes surrounded by a dense ECM consisting of collagen,proteoglycans and water. Several types of cartilage are recognized inthe art, including, for example, hyaline cartilage, articular cartilage,costal cartilage, fibrous cartilage, meniscal cartilage, elasticcartilage, auricular cartilage, and yellow cartilage. The production ofany type of cartilage is intended to fall within the scope of theinvention. According to an embodiment, the invention is directed tocompositions and methods for the production of new cartilage tissue foruse preferably in humans. However, the invention may also be practicedso as to produce new cartilage tissue for use in any mammal in needthereof, including horses, dogs, cats, sheep, pigs, among others. Thetreatment of such animals is intended to fall within the scope of theinvention.

The term “tendon tissue” is used herein as that term is generallyrecognized in the art, and refers to a specialized type of fibrousconnective tissue, comprising mainly collagen, proteoglycans and water.

The term “skin tissue” is used herein as that term is generallyrecognized in the art, and refers to a specialized type of fibroustissue also comprising collagen, elasitin, and proteoglycans.

In one embodiment of the present invention, it is disclosed anon-enzymatic method to liberate mechanically and rapidly early adherentcell populations that define the characteristics of a parental cell bank(PCB) establishment. In some embodiments, the primary, differentiatedcells come from specific cartilage tissue, from specific tendon tissueand from specific skin tissue. The development of the PCB allowsconsistent and stable further cell banking to be accomplished.

Thus according to an embodiment of the present invention, it is providedan in vitro non-enzymatic method for isolation, expansion anddevelopment of foetal cells selected from the group consisting of foetalepiphyseal chondrocytes, foetal Achilles tenocytes or foetal skinfibroblsts, comprising the steps of:

-   -   a) using foetal sample selected from ulnar foetal cartilage        comprising foetal epiphyseal chondrocyte; foetal Achilles tendon        comprising foetal Achilles tenocytes; or foetal abdominal skin        comprising foetal skin fibroblasts;    -   b) micro-dissecting and dispersing said ulnar foetal cartilage,        foetal Achilles tendon or foetal abdominal skin sample by        mechanical attachment to scalpel scored surface;    -   c) culturing said ulnar foetal cartilage, foetal Achilles tendon        or foetal abdominal skin sample in vitro under conditions        wherein said foetal epiphyseal chondrocytes, foetal Achilles        tenocytes or foetal abdominal skin fibroblasts proliferate,    -   d) selecting and isolating first adherent foetal epiphyseal        chondrocyte cell populations, first adherent foetal Achilles        tenocyte cell populations and first adherent foetal skin        fibroblast populations therefrom.

Preferably ulnar foetal cartilage sample is a sample of foetal proximalulnar epiphysis.

In a specific embodiment, the invention relates to a method for theisolation, expansion and development of a foetal epiphyseal chondrocyteor chondro-progenitor (FEC), foetal Achilles tendon progenitor, andfoetal skin progenitor parental cell banks from a single tissue donation(only 0.2-2 cm tissue). The source of cartilage, tendon and skin isimportant to establish consistent cell banks Applicants have found thatulnar foetal cartilage is a superior source than from tibia, femur orrib cartilage, that foetal Achilles tendon is superior and foetal skinfrom abdomen is superior. Foetal cartilage, tendon and skin haveremarkable abilities for repair and foetal cells exhibitimmune-modulatory activity and significant wound healing capabilities.For cartilage, these aspects, in combination with their naturalosteochondrogenic ability following epiphyseal ossification and thecellular characterization of FECs make this population of cells isolatedin non-enzymatic method of the present invention a very interestingcellular choice for osteochondral and/or cartilage tissue repair andregeneration.

Traditional primary culture methods of digestion for liberation of thecell population of choice are not used in the method of the invention.Digestion is made generally from tissues that do not dissociateautomatically (i.e. blood cellular components, some placenta tissuesections, some umbilical cord sections). Mechanical dissection of tissuecombined with directed tissue/cell growth along incisions into thetissue culture plates, selects the first adherent cell population inseveral days of growth only and this population is very consistent andhomogenous. These cell populations grow more rapidly and have differentmorphological and physiological profiles from other cell populationsthat have been digested enzymatically. Applicant has shown growthdifferences in 2D for foetal skin tissue which has been enzymaticallydigested compared to mechanical treatment with cell alignment onsurface.

Importantly, if the primary cultures are developed without digestion oftissue, the foetal articular chondrocytes, foetal tendon progenitors andfoetal skin progenitors cannot de-differentiate into other cell lineageseasily. Chondrocytes or chondro-progenitors, tendon progenitors and skinprogenitors developed without enzymatic digestion in the cell bankingprocedure do not differentiate into neural, adipogenic and fullosteogenic cells such as mesenchymal stem cells derived from bone marrowor from foetal articular cartilage cells that have been treatedenzymatically for their primary cell culture or that do not have solidtissue components to attach to plastic culture dishes. Another importantaspect is that the developed cell banks from the non-enzymatic cellprimary cell culture results in cells that are more stable over passagesin vitro. Morphology and chromosome stability are important factors forusing these banked cells for therapeutic agents in humans.

The thorough cGMP isolation using a non-enzymatice method and processingcombined with the observed homogeneity and stability of this FECpopulation, the tendon progenitor population and the skin progenitorpopulation in culture allows for the reliable expansion of FECs, tendonprogenitors and skin progenitors for in vitro and development ofextensive Master Cell Banks from the PCB population of this describedmethod. It also makes it possible to use the same cell bank of eachtissue for clinical applications of hundreds of thousands of patients,opening the door to novel osteochondral, musculoskeletal and skinregeneration therapies.

Musculoskeletal tissues, such as tendon, bone, muscle, disc andcartilage, and skin tissues when derived from fetal tissues of 10-16weeks are ideal sources to develop parental cell banks rapidly forclinical use. The cells derived when the tissue is not enzymaticallydigested and when cell alignment is directed along serrated surfaces,are prepared rapidly (within 12-14 days versus several weeks to monthswhen the tissue is subjected to enzymatic digestion), are uniform inpopulation and maintain characteristics of specific tissue-to-cell typewith associated bio-markers.

The non-enzymatic method of the present invention provides differentcells than those obtained by known enzymatic methods. Thus in anembodiment, the present invention provides a foetal epiphysealchondrocyte cell line, obtained by the non-enzymatic method of thepresent invention, having the designation FE002-Cart and deposited underaccession number ECACC 12070303-FE002-Cart on 3 Jul. 2012. In anotherembodiment, the present invention provides foetal Achilles tenocyte cellline (also denoted herein as foetal Achilles tendon progenitors cellline), obtained by the non-enzymatic method of the present invention,having the designation FE002-Ten and deposited under accession numberECACC 12070302-FE002-Ten on 3 Jul. 2012. In a further embodiment, thepresent invention provides foetal skin fibroblast cell line (alsodenoted herein as foetal skin progenitors cell line), obtained by thenon-enzymatic method of the present invention, having the designationFE002-SK2 and deposited under accession number ECACC 12070301-FE002-SK2on 3 Jul. 2012.

Once established, a culture of foetal epiphyseal chondrocytes orchondro-progenitors may be used to produce chondrocytes capable ofproducing new cartilage cells and tissue. Differentiation of foetalepiphyseal chondrocyte to chondrocytes, followed by the production ofcartilage tissue therefrom, can be triggered by the addition to theculture medium with or without the use of specific exogenous growthfactors, such as, for example, BMPs such as BMP-13 or TGF-β, with orwithout ascorbate. The same procedures from established foetal tendonprogenitors and foetal skin progenitors can be applied.

The invention further contemplates the establishment and maintenance ofcultures of chondrocytes as well as mixed cultures comprising bothfoetal epiphyseal chondrocyte and chondrocytes. As with foetalepiphyseal chondrocyte, once a culture of chondrocytes or a mixedculture of foetal epiphyseal chondrocyte and chondrocytes isestablished, the population of cells is mitotically expanded in vitro bypassage to fresh medium as cell density dictates, under conditionsconducive to cell proliferation without cartilage formation, such as,for example, in culture medium lacking TGF-β or other growth factor. Aswith cultures of pre-chondrocytes, cultures of chondrocytes and mixedcultures of foetal epiphyseal chondrocyte and chondrocytes should betransferred to fresh medium when sufficient cell density is reached.Thus, formation of a monolayer of cells should be prevented orminimized, for example, by transferring a portion of the cells to a newculture vessel and into fresh medium. Such removal or transfer should bedone in any culture vessel which has a cellular monolayer exceedingabout 25% confluency. Alternatively, the culture system can be agitatedto prevent the cells from sticking. The same method can be equallyapplied for foetal tendon progenitors and foetal skin progenitors.

In a further embodiment of the invention, a population of foetalepiphyseal chondrocytes or chondro-progenitors isolated from ulnarfoetal cartilage is mitotically expanded and cultured in vitro to giverise to chondrocytes which can produce cartilage tissue and cells fortherapeutic use. The same method can be equally applied for foetaltendon progenitors and foetal skin progenitors. Thus in an embodiment,the present invention relates to foetal epiphyseal chondrocyte (FEC),foetal Achilles tenocytes and foetal skin fibroblasts obtained by thenon-enzymatic method of the invention for use as therapeutic agents.

In another embodiment of the invention, foetal epiphyseal chondrocytesor chondro-progenitors isolated from ulnar foetal cartilage, arecryopreserved and stored frozen in a “bank” from which they can bethawed and used to produce cartilage tissue and cells as needed. Thefoetal epiphyseal chondrocytes or chondro-progenitors, which areharvested or produced therefrom, can be stored frozen in a “bank” for aperiod of years. The cells may be withdrawn from the bank as needed bythawing, and the thawed cells can be used to produce new tissues andcells at any time for the repair, replacement or augmentation ofcartilage, as well as other mesenchymal tissues, such as bone, tendon orligament. The same method can be equally applied for foetal tendonprogenitors and foetal skin progenitors.

As a result of the “foetal” nature of the cells isolated from ulnarfoetal cartilage sample, immune rejection of the implanted foetalepiphyseal chondrocyte of the invention, or cartilage tissue producedtherefrom, may be minimized. Accordingly, in another embodiment of theinvention, such cells are useful as “ubiquitous donor cells” for use inany subject in need thereof. The same characteristics can be equallyseen for foetal tendon progenitors and foetal skin progenitors.

In another embodiment of the invention, the foetal epiphysealchondrocyte or chondro-progenitor cells, foetal Achilles tendonprogenitors or foetal skin progenitors are suspended in a hydrogelsolution where they can be either injected or implanted into a patient.Alternatively, the cells may be first seeded into the hydrogel, and thencultured prior to implantation. Preferably, the cells are cultured inthe hydrogel so that they mitotically expand prior to implantation.

In yet another embodiment of the invention, new cartilage tissue andcell populations are prepared from the foetal epiphyseal chondrocyte orchondro-progenitor cells of the invention and is used to repair, replaceor augment cartilage tissue in a subject using any technique of repair,replacement or augmentation known in the art or to be developed in thefuture. For example, the foetal epiphyseal chondrocyte orchondro-progenitors cells of the invention may be seeded onto athree-dimensional framework or scaffold composed of a biocompatiblenon-living material having interstitial spaces, openings or pores thatcan be bridged by the chondrocytes. Under appropriate in vitro cultureconditions, the seeded cells substantially envelope thethree-dimensional framework and secrete an extracellular matrix to forma new, living cartilage tissue which can be implanted in vivo.Alternatively, the foetal epiphyseal chondrocyte or chondro-progenitorcells of the invention are seeded onto a three-dimensional framework andimmediately implanted at a site in the subject. The seeded cells proceedto form new cartilage tissue in vivo or stimulate receiver cartilage torepair and re-organize. The same method can be equally applied forfoetal tendon progenitors and foetal skin progenitors in the means toform or stimulate new tendon tissue or new skin tissue of the receivertendon or skin to repair and re-organize.

In yet another embodiment, the three-dimensional framework on which thecells of the invention are seeded further comprises, or is coated with,one or more bioactive agents or other compounds selected from the groupconsisting of anti-inflammatories, growth factors, immunosuppressants,etc.

In yet another embodiment of the invention, the foetal epiphysealchondrocyte or chondro-progenitor cells of the invention are inoculatedand grown on a three-dimensional framework and placed in a containerthat can be manipulated to allow intermittent pressure changes, or in abioreactor system specially designed for the in vitro production ofcartilage tissue constructs, which bioreactor allows for pressurizationof the chamber during growth and an adequate supply of nutrients tochondrocytes by convection. The same method can be equally applied forfoetal tendon progenitors and foetal skin progenitors.

Scaffold based cell therapy provides a palpable advantage in that thetherapeutic tissue-generating agent delivered, in this case the cells,are easily localized and can therefore be implanted arthroscopicallyalong with the scaffold (Iwasa et al., 2009). This bypasses the need formajor invasive surgery (total joint replacement) and preserves thenative tissue as best as possible, thereby significantly reducing theoccurrence of inflammation, which may very well negatively affect theoutcome of the therapy (van Osch et al., 2009). In order to provide atemplate supporting 3D tissue growth, one has to carefully tailor ascaffold's structure and composition. Synthetic materials such aspolyethylene glycol (PEG), polylactic acid (PLA) and polyglycolic acid(PGA) as well as naturally derived materials such as hyaluronan,chondroitin sulfate and chitosan have been used with or without furtherchemical modification and side group addition in order to generate boneand cartilage (Ahmed et al., 2010; Chung et al., 2008; Khan et al.,2008).

Biomaterial scaffolds function as the extracellular matrix to provide aphysical structure to protect cells and to guide tissue growth.Integration into the matrix is essential to have a three-dimensionalsystem for delivery to the surgical site of interest. Foetal cells,unlike adult and mesenchymal cells, have been shown to penetratethroughout various biomaterials due to their inherent adhesion andmigration properties. This allows for rapid seeding of the cells intothe respective biomaterials and less manipulation of the implants beforeimplantation.

Different materials and fabrication methods can form biomaterialscaffolds with distinct properties which can adapt to cartilage tissueengineering. Many biodegradable biomaterials and hydrogels have beendeveloped for other surgical purposes such as hemostatic sponges andtissue fillers (Mast et al., 1993; Patino et al. 2002; Drury and Moony,2003). These biomaterials provide clinical-grade materials (classifiedas Medical Devices) to be tested for biocompatibility and to assure thatthere are no radical derivative products produced by the association ofthe biomaterial delivery system and the cellular products.

In a further embodiment, the foetal epiphyseal chondrocyte orchondro-progenitor cells of the invention are administered directly to asite in vivo, e.g., by injection and without attachment to athree-dimensional framework, to produce new cartilage tissue and cellpopulations at that site. The same method can be equally applied forfoetal tendon progenitors and foetal skin progenitors. Thus in anembodiment, the present invention relates to use of foetal epiphysealchondrocyte (FEC) obtained by the non-enzymatic method of the inventionfor the production of new cartilage tissue and/or three dimensionalconstructs; use of foetal Achilles tenocytes obtained by thenon-enzymatic method of the invention for the production of new tendontissue and/or three dimensional constructs and use of foetal skinfibroblasts obtained by the non-enzymatic method of the invention forthe production of new skin tissue and/or three dimensional constructs.

In a further embodiment of the invention, the foetal epiphysealchondrocyte or chondro-progenitor cells of the invention are stimulatedto produce cartilage using exogenously supplied growth factors such as,for example, TGF-β or BMPs such as BMP-2, BMP-12 and BMP-13.

In yet another embodiment of the invention, the foetal epiphysealchondrocyte or chondro-progenitor cells of the invention can begenetically engineered to produce, or to increase production of,specific types of growth factors, peptides, proteins or other moleculesthat serve to increase the amount of cartilage produced, or that improvethe success of implantation, for example, by reducing the risk ofrejection or inflammation associated with the implant. The same methodcan be equally applied for foetal tendon progenitors and foetal skinprogenitors.

The invention also relates to the products of the foregoing methods,including but not limited to, the foetal epiphyseal chondrocyte orchondro-progenitor cells of the invention, mitotically expanded orotherwise; new cartilage tissue produced therefrom; extracellular matrixextracted therefrom; and three-dimensional cartilage/frameworkconstructs. The invention also relates to the use of these cells,constructs and tissues in vivo to repair, replace or augment cartilage,or in vitro to form three-dimensional cartilage cultures which areuseful to produce new cartilage tissue or bioactive agents, or to testthe cytotoxicity of potential therapeutic agents. The same method can beequally applied for foetal tendon progenitors and foetal skinprogenitors.

In an embodiment of the present invention, the foetal epiphysealchondrocytes or chondro-progenitors isolated from ulnar foetalcartilage, as well as the chondrocytes differentiated therefrom, can beused to produce new cartilage tissue and cells to repair or replacecartilage. The same method can be equally applied for foetal tendonprogenitors and foetal skin progenitors which can be used to produce newtendon or skin tissues and cells to repair or replace tendon or skin.

In a further embodiment of the present invention, the foetal epiphysealchondrocytes or chondro-progenitors isolated from ulnar foetal cartilageaccording to the method of the present invention, as well as thechondrocytes differentiated therefrom, can be used as therapeutic agent.Preferably, the foetal epiphyseal chondrocyte isolated from ulnar foetalcartilage according to the method of the present invention, as well asthe chondrocytes differentiated therefrom, are for use in the method forosteochondral repair (including cartilage repair and tendon repair) andregeneration, and in method for treating osteochondral diseases ordefects (including osteochondral lesions, injuries, trauma,fragmentations and fractures, subchondral bone necrosis andosteochondritis) and/or arthritis. The same method can be equallyapplied for foetal tendon progenitors and foetal skin progenitors forspecific treatments of tendon and skin.

Thus in an embodiment, the present invention relates to foetalepiphyseal chondrocyte (FEC) obtained by the non-enzymatic method of theinvention for use in a method for repair and regeneration ofosteochondral tissue and musculoskeletal tissue; foetal Achillestenocytes obtained by the non-enzymatic method of the invention for usein a method for repair and regeneration of tendon tissue andmusculoskeletal tissue; and foetal skin fibroblasts obtained by thenon-enzymatic method of the invention for use in a method for repair andregeneration of skin tissue and for treating burns, wounds and fibroticcondition.

In a further embodiment of the present invention, is provided foetalepiphyseal chondrocyte (FEC) obtained by the non-enzymatic method of theinvention for use in a method for treating osteochondral diseases ordefects, arthritis and musculoskeletal diseases; foetal Achillestenocytes obtained by the non-enzymatic method of the invention for usein a method for treating musculoskeletal diseases and tendonopathies;foetal skin fibroblasts obtained by the non-enzymatic method of theinvention for use in a method for treating skin diseases.

The use of foetal epiphyseal chondrocytes (FEC) or chondro-progenitorsfor osteochondral tissue engineering is very promising. Indeed,throughout foetal development, the epiphysis becomes the major site forthe secondary ossification centre (SOC), which transforms the FECcondensate into both an articular cartilaginous region and what becomesepiphyseal trabecular bone and marrow after vascular invasion (Blumer etal., 2008; Onyekwelu et al., 2009). It is known that foetal cartilage,such as that seen for foetal skin, has a remarkable capability forself-repair. In a foetal goat model, defects have been shown to repairwith out scar or fibrous tissue formation (Namba et al., 1998).

The cells (foetal epiphyseal chondrocytes or chondro-progenitors andchondrocytes) and cartilage tissues of the invention may be used invitro to screen a wide variety of compounds for effectiveness andcytotoxicity of pharmaceutical agents, growth/regulatory factors,anti-inflammatory agents, etc. To this end, the cells of the invention,or tissue cultures described above, are maintained in vitro and exposedto the compound to be tested. The activity of a cytotoxic compound canbe measured by its ability to damage or kill cells in culture. This mayreadily be assessed by vital staining techniques. The effect ofgrowth/regulatory factors may be assessed by analyzing the number ofliving cells in vitro, e.g., by total cell counts, and differential cellcounts. This may be accomplished using standard cytological and/orhistological techniques, including the use of immunocytochemicaltechniques employing antibodies that define type-specific cellularantigens. The effect of various drugs on the cells of the inventioneither in suspension culture or in the three-dimensional systemdescribed above may be assessed. Thus the foetal epiphyseal chondrocytesor chondro-progenitors isolated from ulnar foetal cartilage can be alsoused to develop advanced therapeutic agents and/or medical device forthe treatments of arthritic, osteochondral defects, cartilage repair,tendon repair. The same method can be equally applied for foetal tendonprogenitors and foetal skin progenitors which can be also used todevelop advanced therapeutic agents and/or medical device for thetreatments of musculoskeletal tissues.

Thus in an embodiment of the present invention, it is provided ascreening method for development of therapeutic agents and/or medicaldevices for the treatment of arthritic, osteochondral defects, cartilagerepair, tendon repair, musculoskeletal tissue repair and skin repair,comprising the use of foetal epiphyseal chondrocyte (FEC), foetalAchilles tenocytes or foetal skin fibroblasts obtained by thenon-enzymatic method of the invention.

The cells (foetal epiphyseal chondrocytes or chondro-progenitors andchondrocytes) and cartilage tissues of the invention may be used asmodel systems for the study of physiological or pathological conditions.For example, joints that are immobilized suffer relatively quickly in anumber of respects. The metabolic activity of chondrocytes appearsaffected as loss of proteoglycans and an increase in water content aresoon observed. The normal white, glistening appearance of the cartilagechanges to a dull, bluish color, and the cartilage thickness is reduced.However, the amount of this change that is due to nutritional deficiencyversus the amount due to upset in the stress-dependent metabolichomeostasis is not yet clear. The cells and cartilage tissues of theinvention may be used to determine the nutritional requirements ofcartilage under different physical conditions, e.g., intermittentpressurization, and by pumping action of nutrient medium into and out ofthe cartilage construct. This may be especially useful in studyingunderlying causes for age-related or injury-related decrease in tensilestrength of articular cartilage, e.g., in the knee, that predispose theweakened cartilage to traumatic damage. The same method can be equallyapplied for foetal tendon progenitors and foetal skin progenitors usefulin studying underlying causes for age-related or injury-related decreasein tensile strength of tendon and skin to traumatic or age-relateddamage.

The cells (foetal epiphyseal chondrocytes or chondro-progenitors andchondrocytes) and cartilage tissues of the invention may also be used tostudy the mechanism of action of cytokines and other pro-inflammatorymediators, e.g., IL-1, TNF and prostaglandins, that are released intothe synovial fluid as a result of rheumatic disease. Thus, the patient'sown joint fluid could be tested in vitro to study the effects of thesecompounds on growth of the cells of the invention. In addition,cytotoxic and/or pharmaceutical agents can be screened for those thatare most efficacious for a particular patient, such as those that reduceor prevent resorption of cartilage or otherwise enhance the balancedgrowth of articular cartilage. Agents which prove to be efficacious invitro could then be used to treat the patient therapeutically. The samemethod can be equally applied for foetal tendon progenitors and foetalskin progenitors useful in studying mechanism of action of growthfactors, cytokines and other pro-inflammatory mediators which cause animbalance in tissue repair.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications without departing fromthe spirit or essential characteristics thereof. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.The present disclosure is therefore to be considered as in all aspectsillustrated and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples are, however, exemplary ofmethods of practising the present invention and are not intended tolimit the scope of the invention.

EXAMPLES

The proximal ulnar epiphysis, the Achilles tendon and abdominal skinwere processed following strict transplantation laws and by-laws andguidelines for organ donation and screening to create FEC, tendon andskin parental cell banks for tissue engineering applications (CHUVEthics Committee protocol #62/07). The tissue biopsies (cartilage, ˜2mm³, tendon, −0.2 mm³, skin, ˜2 cm²) were micro-dissected and dispersedby mechanical attachment to scalpel scored surfaces. (No enzymatictreatments were used to assure only adherent cartilage, tendon or skinoutgrowth and consistency of cell population: necessary criteria forclinical usage). FEC, tendon and skin outgrowth was observed at 1-2 daysto one week and expansion was accomplished at one and at two weeks. Theparental cell bank was established with 50-200 vials of 5-10×10⁶ cellsand stored in the vapour-phase of liquid nitrogen. In vitrocharacterization of isolated cells exhibit a remarkable homogeneity inmonolayer culture as well as a notable proliferative potential with apropensity for overlapping (3D culture). FECs, tendon progenitors andskin progenitors do not seem to exhibit notable phenotypic variationswithin the first six passages. FEC and tendon progenitors are able tospontaneously coalesce when placed in pellet culture form, depositmatrix and express fundamental chondrogenic or tenocyte markers. Flowcytometric analyses revealed unimodal distributions indicative of ahomogenous population. A comparison with adult bone marrow derived MSCsyielded FEC or tendon surface marker profiles consistent with achondrogenic or tendocyte rather than an undifferentiated progenitorphenotype.

Foetal Chondrocyte, Tendon Progenitor and Skin Progenitor Cell Banking,Characterization in Relation to Therapeutic Agent Preparation

Cell Banking

Cell banks have been established in the University Hospital of Lausannefrom foetal biopsies between 12-14 weeks of gestation obtained afterpregnancy termination with informed and written consent and approvalfrom the local Medical School Ethics Committee since 1993 and moreparticularly for clinical cell banks since 2007. Pre-clinical articularcartilage cell banks have been successfully developed from 2 independentdonors to date. Using one of these (FE002-Cart p.0, FE-002-Ten p.0,FE-002-SK2, p.0), it will be possible to establish all of the cellsnecessary for pre-clinical and clinical trials at low passages (MCB atp.3 and WCB at p.5).

From ˜0.3 cm³ articular cartilage (radius) (see FIG. 7), from ˜0.2 mm³Achilles tendon and from ˜1 cm² abdominal skin, pre-clinical cell bankswere prepared at passages 0 and 1. Tissue was dissected into <0.5 mm³fragments, where possible, and grown in DMEM supplemented with 10% FCSand glutamine and the cells were used for characterization andexperimentation at passage 3 and 6 or 1 and 9. They were grown toconfluence before splitting and rinsed twice with PBS and counted.

Detailed Procedure Without Enzymatic Processing

The tissue was divided into two, 10 cm plates with whole tissuefragments ˜5 per plate (<0.5 mm³), where possible. Tissue culture disheswere previously scored deeply with a sterile scalpel in a check-boardpattern under the laminar flow hood. Tissue fragments were placed intothe scored plastic regions mincing gently and attaching the fragmentswithin the plastic indentions. A small quantity of nutrient media wasplaced around each fragment to avoid floating of tissues for the first24 hours. Following the first 24 hours, 8 ml of culture media was addedonto each 10 cm plate and this was changed two times per week beforepassage. These fragments were grown in DMEM supplemented with only 10%foetal bovine serum (Hyclone) to help insure a consistent cell culture.Cell cultures were grown at 37° C. in a humidified atmosphere of 95%air/10% CO₂. It is important to mention that any nutrient componentnecessary for cell culture for clinical trials should have thoroughsafety requirements and tracing. All animal derived products, such asfor foetal bovine serum and trypsin, specific clinical lots of trypsinand gamma irradiated serum that have been tested for adventitious agentsshould be employed. Cell growth was first seen even following one daybut after cell growth advanced during 5-7 days, dishes of tissue andcells were removed from plates either by trypsinizing (0.25%trypsin-0.1% ethylene diaminetetraacetic acid [EDTA]) or with EDTA aloneto passage to multiple tissue culture flasks or frozen for cell banking.At this point, some foetal cartilage, foetal tendon or foetal skin cellswere frozen into individual units in liquid nitrogen and others werepassaged at 1,000-2,000 cells/cm² or 10,000-50,000 cells/cm² forproducing the PCB. Cells were centrifuged at 2000 g for 15 min andresuspended in a freezing solution of DMEM (5 ml)+FCS (4 ml)+DMSO (1 ml,Fluka) and frozen in 1 ml aliquots (˜5-10 million cells) at −80° C. inNalgene Cryo 1° C. Freezing Container's (Nalgene) to achieve a −1°C./min rate of cooling and freezing curve. After 24 hr, cells weretransferred to liquid nitrogen for longer storage.

1-2 vials of pre-clinical foetal articular cartilage, foetal tendon orfetal skin are used to prepare a consistent Working Cell Bank (WCB) forcharacterization. Procedure design is the same as that used in cGMPmanufacturing. In short, cells are initiated at 1,500 cells/cm², or from1,000-100,000 cells/cm² into 100 cell culture flasks (T75, Nunc) with 15ml of nutrient media (DMEM+10% clinical grade serum, foetal bovine,Invitrogen). Cells have the media changed every 2 days and theproduction/expansion of cells is done for 10-14 days at 37° C., 10%humidity or for 3-6 days at higher density seedings. At day 12-14,batches of 10 T75 flasks are subjected to TrypLE to separate the cellsfrom the flasks and placed into centrifuge tubes. An equal volume ofnutrient media is added to each tube before centrifugation to bufferTrypLE effects. The cell pellet obtained from all of the flasks areresuspended into 200 ml freezing media solution (DMEM, Serum, DMSO,5:4:1 ratio) and aliquoted into 100 Nunc freezing viles (1.5 ml) at adilution of 10×10⁶ cells/ml.

Cells are frozen at −80° C. in Nalgene Cryo 1° C. Freezing Container's(Nalgene) to achieve a −1° C./min rate of cooling and freezing curve.After 24 hr, cells are transferred to liquid nitrogen in the gas phase.This WCB is at passage 2 and used for cell characterization and cellsare used at various passages but mainly between 2-8.

Foetal Chondrocyte Characterization: Identity, Stability, Consistency,Genetic Stability

Banked foetal chondroytes have been compared throughout Applicants'study to BM-MSC cells that have been banked under the same technicalconditions in their lab. MSC's are allogenic cells and one of the mainother cell type that have been proposed and used in pre-clinical andclinical experimentation for cartilage regeneration among other tissues(however, mesenchymal cells are not tissue specific and have to beprogrammed to make other tissue types). Moreover, MSC's are moreinstable and have to be used under passage 4. The same method can beequally applied for foetal tendon progenitors and foetal skinprogenitors

Foetal Cartilage Characterization Compared to BM-MSC

Foetal articular cartilage cell banks have been characterized by surfacemarkers accomplished with FACS analysis, functional assays for matrixdeposition compared to BM-MSC cells. Foetal cartilage cells derived fromarticular epipheseal tissue have been isolated and parental cell bankshave been frozen.

Some markers that have been compared in preliminary experiments include:

-   -   CD105: Endoglin. Part of TGFBeta receptor complex. Main        criterion for MSC positive selection.    -   CD90: Thy-1. Main criterion for MSC positive selection.    -   CD44: PTPRC. Leukocyte marker. Criterion for negative selection        of bmMSCs.    -   CD73: NT5E. Main criterion for MSC positive selection.    -   CD166: ALCAM.        (See FIGS. 8 and 9)

The same method can be equally applied for foetal tendon progenitors andfoetal skin progenitors

Biocompatibility: Foetal Cartilage Biocompatibility with Hydrogels andMatrix

Different matrixes that are available for medical use are initiallytested for biocompatibility. Hydrogels of various compositions,collagens and some biodegradable polymers are used in the firstexperiments. For hydrogels, cells are cultivated within the gel that isinserted into a pre-prepared agar mold. This is necessary to avoidcellular attachment to the tube and to allow three dimensional growth.The mold is prepared by pipetting 1 ml of melted agarose (20% agar,low-melting agar) inside a 1.5 ml sterile conical eppendorf centrifugetube for which a 0.5 ml sterile conical eppendorf centrifuge tube isinserted into the liquid agar and allow to solidify before extractingthe 0.5 ml tube leaving a conical inset. Following gel and celladdition, 100 μl media is pipetted onto the surface of each tube andmedia changed two times weekly. Cells are grown for one, two and fourweeks in a 37° C. incubator at 95% relative humidity and 10% CO₂.

For matrix preparations, preliminary experiments investigating cellseeding density (from 10³ to 10⁴ cells cm²) and growth periods (from 1to 28 days) are performed in order to determine optimal conditions forfoetal cell delivery. Foetal cells and BM-MSC's at passages 3 or 4(maximum 4 for MSC due to instability thereafter) are placed in 10 mlmedia (DMEM containing 10% FBS) and seeded on matrix. The matrixcontaining the cells is placed into a 37° C. incubator at 95% relativehumidity and 10% CO₂. An additional 30 ml media is then added one hourlater. Matrix is changed twice weekly with nutrient media.Biocompatibility is also measured by a contact assay where cells arecultured within a tissue culture plate where hydrogel or matrix wereinitially seeded. Cell growth and migration are analysed visually withrespect to hydrogel and matrix interface. Following one, two and fourweeks, samples are stained with giemsa and photographed (Sony CyberShotDSC-570, Zeiss macro lens, Zoom 6×, 3.3 megapixels).

The invention claimed is:
 1. An in vitro non-enzymatic method forisolation, expansion and development of fetal cells selected from thegroup consisting of fetal epiphyseal chondrocytes, fetal Achillestenocytes and fetal skin fibroblasts, comprising the steps of: a)obtaining a fetal tissue sample selected from the group consisting ofulnar fetal cartilage comprising fetal epiphyseal chondrocytes; fetalAchilles tendon comprising fetal Achilles tenocytes; and fetal abdominalskin comprising fetal skin fibroblasts; b) micro-dissecting said fetaltissue sample; c) dispersing said fetal tissue sample by mechanicalattachment to a scalpel scored surface within an indention; d) culturingsaid fetal tissue sample in vitro under conditions wherein said fetalepiphyseal chondrocytes, fetal Achilles tenocytes or fetal abdominalskin fibroblasts proliferate; and e) selecting and isolating a firstadherent fetal epiphyseal chondrocyte cell population, a first adherentfetal Achilles tenocyte cell population, or a first adherent fetal skinfibroblast population therefrom, wherein said selecting step isperformed 5 to 7 days post-dissection.
 2. The method of claim 1, whereinsaid ulnar fetal cartilage tissue sample is a sample of fetal proximalulnar epiphysis.